Acta histochem, Bd. 61, S. 1-19 (1978)

Biological-Morphological Institute, Silesian Medical School,

Head of the Institute: Prof. Dr. J. J. JONEK, M. D.,Department of Cytology and Histology,

Head of the Separtment: Prof. Dr. J. J. JONEK, M. D.

Histochemical and histoenzymatic changes in mouse liverin subacute benzene intoxication

By MARCIN KAMINSKI, J. JAN JaNEK, JANUSZ KONECKI,OLGA KAMIN-SKA, BARBARA GRUSZECZKA and BRIGIDA KOEHLER

With 38 figures

(Received May 15, 1977)

Summary

The investigations were performed on mice. They were divided into a control group and 4 ex­

perimental groups. The experimental animals were administered intraperitoneally benzene 6 Xevery 24 h. The animals were decapitated 30 min, 4, 12 and 24 h after the last benzene administra­

tion. During the experiment, dyeing for neutral lipids and glycogen was carried out, and the ac­tivity of NADH2·r.t., SDH, G-6-P-ase, A'I'Pvase and ACP was estimated.

A decrease of glycogen content in liver cells, deviations in the amount of neutral lipids, re­versible decrease of mitochondrial enzymes activity, and intensification of the processes of intra­cellular catabolism were found.

The glandular cells of the liver are mainly responsible for proper and efficientprocess of intraconstitutional benzene conversion. In this connection, hepatocyteshave various enzymes that carry out oxygenation of this substance to phenol, catechol,and 2-hydroxy-chinon (1, 5, 6, 12, 18, 19). Phenol can be oxidized to hydroxychinon,or undergo conjugation with sulphuric and glycuronic acid under the influence ofsulphate and glycuronide enzymes (1, 18).

In extreme cases increased oxidizing processes can lead to an opening of the

aromatic ring and production of muconic and phenylmercapturic acid. Various waysof benzene conversion that takes place in the liver show that the liver is a very im­

portant organ in the processes of detoxication, as well as provides support that theorganism is resistent to harmful action of toxic substances. The aim of this work

was to study the pathomechanism of the influence of benzene and its metabolitesupon the liver.

Material and Methods

The experiments were carried out on 52 sexually mature, inbred mice of Porton Strain weighing

32 ± 1,5 g. The animals were divided into 4 experimental groups - each containing 10 animals,and 1 control group.

1 Acta histochem. Bd, 61

2 M. KAMINSKI et al,

Benzene was administered intraperitoneally to experimental animals 6 times every 24 h in thedose of 1000 mgjkg of body weight in the volume of 0,03 m!. The animals of each particular groupwere decapitated 30 min, 4, 12 and 24 h after the administration of the 6th dosage of benzene.

Control animals received peritoneally 0,03 ml of physiological solution, 6 X in every 24 h, and

3 mice of each experimental group were killed. At autopsy, liver samples 3 mm thick were always

taken from the same region of the left lobe. The obtained material was divided into 3 parts. One

of them was kept in a cryostat, cooled to - 20 "C, and then cut into slices 10 flm thick. After

sticking them onto the egg-albuminized base glasses the reactions were made for:

1. Succinic dehydrogenase (SDH) using Tetra Nitro BT produced by Sigma according toPEARSE (16). The incubation time was 4 min at 37 "C.

2. Glucose-6-phosphatase (G-6-P-ase) with glucose-6-phosphate produced by Sigma accordingto PEARSE (16). The incubation time was 12 min at 37 "C.

The other part of the material was fixed for 12 h in cold (4 "C) BAKER'S solution, and thencut in a freezing microtome into sections 10 flm thick. After sticking them on albuminized baseglasses, the following reactions were made:

1. NADH2-tetrazole reductase (NADH2-t.r.) using Nitro BT produced by Sigma according toFARBER (3). The incubation time was 40 min at 37 "C. At the same time an acetone test was car­

ried out.

2. Adenosine triphosphatase (ATP-ase) Ca formol using bisodium salt ATP as substrate

produced by Koch-Light, aceording to WACHSTEIN and MEISEL (20). The incubation time was20 min at 37 "O,

3. Acid phosphatase (ACP) using sodium glycerophosphate produced by BDH by means of

GOMOR! method (4). The incubation time was 60 min at 37 "C.

4. Staining of neutral fats on freely floating segments with oil Red-O produced by Michromeaccording to BAGINSKI.

The 3rd part of the material was fixed in GENDRE'S fluid. Sections 10 flm thick were prepared,and PAS reaction for glycogen according to MACMANUS (13) was carried out using diastase di­gestion of glycogen.

Results

Staining for neutral fats with Oil Red-o method

Control group: The colour reaction for neutral fats in the liver cells of healthyanimals was weak. In the lobules it was localized in the region of the central zone(middle vein) and around the vessels of the lobule circumference (Fig. 1). Few, small,bright red drops arranged in the cytoplasm of hepatocytes, usually irregular, oftennear the membrane of the nucleus were observed in these cells. In the remainingzones of the lobule the reaction was negative.

Fig.!. Reaction for neutral lipids in the liver lobule of experimental animals. X 100.

Fig. 2. A marked increase of intensity of colour reaction for neutral lipids in the liver lobule.

II experimental group. X 100.

Fig. 3. A decrease in the intensity of colour reaction for neutral lipids in the peripheral zone of

the lobule. III experimental group. X 100.

Fig. 4. Normalization of the reaction for neutral lipids with regard to intensity, character andlocalization in the liver. IV experimental group. X 100.

l'

Histochemical and histoenzymatic changes

Fig. 1-4

3

4 1\'[. KAMINSKI et al.

Experimental group I (30 min): In this group a slight increase of the colourreaction for neutral fats in the liver lobules was observed. This increase particularlyrefers to the central zone. The cells in this zone showed large amount of bigger andsmaller drops containing the reaction product, and seemed to be strongly saturatedwith it.

Experimental group II (4 h): An increase of neutral fats contents was ob­served in the liver lobules. The glandular cells of all zones of the liver lobule were

filled with very numerous and big and strongly saturated drops of the product ofthe colour reaction (Fig. 2).

Experimental group III (12 h): In this group, colour reaction in the liverlobule was displaced to such an extent that its peripheral zone was practically devoidof the positive reaction (Fig. 3). In intermediate and middle zones, the character,localization and intensity of the colour reaction were similar to the previous group.

Experimental group IV (24 h): The character, localization and intensity ofthereaction for neutral fats in the liver lobule 24 h after the last intraperitoneal in­jection were similar to the animals of the control group (Fig. 4).

PAS reaction for glycogen

Control group: Positive reaction for glycogen was found in all zones of theliver lobules of healthy animals. The strongest colour reaction occured in the cellslying near the central vein, where the grains of the product of histoenzymatic reac­tion filled the cytoplasm of hepatocytes (Fig. 5). In the remaining parts of the lobulethe reaction was slightly weaker, and the character and localization were similar.

Experimental group I (30 min): In this group a slight decrease of the reac­tion for glycogen was observed, expecially in the peripheral and intermediate zonesof the lobules. Hepatocytes containing glycogen in the amount similar to that of thecontrol group, and few cells showing very weak reaction were found in these areas(Fig. 6). The localization and character of the reaction in this group were similar tothose observed in the group of healthy animals.

Experimental group II (4 h): Numerous cells of peripheral, intermediate andeven middle zones of the lobules, showed very weak or even negative reaction (Fig. 7).

Fig. 5. Positive fine-grained reaction for glycogen in the middle zone of the liver of the controls.

X 400.

Fig. 6. Cells with a strong and weak reaction for glycogen in the peripheral zone of the liver lobule.I experimental group. X 400.

Fig. 7. A further increase of irregularity in the intensity of colour reaction for glycogen amongadenocytes of the lobule periphery. II experimental group. X 400.

Fig. 8. Very numerous liver cells with no vositive PAS reaction for glycogen in the middle zoneof the lobule accompannied by single hepatocytes filled with strong colour reaction. III experi­mental group. X 400.

Histochemical and histoenzymatic changes

Fig. 5-8

8

5

6 M. KAMINSKI et al.

The liver cells with the intensity, character and localization of the reaction similar tothe control were surrounded by a number of other hepatocytes devoid of colour

reaction.

Experimental group III (12 h): In the peripheral and intermediate zones thenumber of liver cells filled with positive product of histoenzymatic reaction wasminimal. However, in the central zone and around the central vein, glandular cellswhose cytoplasm was filled with positive grain reaction, as well as whole fragments

of trabecules devoid of positive reaction for glycogen could be observed (Fig. 8).

Experimental group IV (24 h): A slight increase in the number of cells havingpositive granular reaction for glycogen was observed in the 3 zones of liver lobules.This increase was particularly significant in the peripheral zone. In the intermediateand central zones around the middle vein of the lobules it was not so marked.

Reaction for NADH-tetrazole reductase (NADH-t.r.)

Control group: Positive histoenzymatic reactions for the examined reductasetook place in all liver lobules. In the majority of lobules the strongest reaction wasobserved in the middle and peripheral zones (Fig. 9). In the glandular cells small

graines were localized evenly in the cytoplasm or near the membrane of the nucleus.

Experimental group I (30 min): In this group a slight decrease in the in­tensity of the enzymatic reaction was observed in the peripheral zones of the lobules,

but in the intermediate and central zones the intensity of the reaction was similarto that of the control group. Besides, glandular cells with strong grain-diffuse reac­tion were mainly found in the immediate vicinity of the vessels of the lobule (Fig. 10).

Experimental group II (4 h): Here, no important differences in character,localization and intensity of the reaction in the liver cells were noticed as comparedwith the control group.

Experimental group III (12 h): In this group the reaction was similar tothat of the previous one, but the grains seemed to be thicker, and were accompaniedby diffuse component (Fig. 11). The changes mentioned above were found to be most

intensive in the peripheral zone of the lobules.

Experimental group IV: In the last of the examined groups in all zones (peri­pheral, intermediate and middle) changes were found in the intensity, character and

Fig. 9. Positive fine-grained reaction for NADH2·tetrazol reductase in liver lobules of the con­

trols. X 240.

Fig. 10. Small number of adenocytes of the liver lobule periphery filled with strong grained­diffuse reaction for NADH2·tetrazol.reductase. I experimental group. X240.

Fig. II. Liver adenocytes with a vositive medium-grained, diffuse enzymatic reaction for NADH2 ­

r.t. III experimental group. X 240.

Fig. 12. Fragments of liver lobules with strong diffuse reaction and adjacent trabecules with

weak fine-grained reaction. IV experimental group. X 240.

Histochemi cal and h ist oenzymatic ch an ges

Fig . 9 -12

7

8 M. KAMINSKI et al.

localization of the reaction for NADH2 of tetrazole reductase. Great focal changesin the liver were noted i.e., fragments of lobules characterized by strong grained­diffuse reaction, and sections of trabecules filled with weak but grained product ofthe enzymatic reaction were observed (Fig. 12). Fragments oflobules filled with stronggrained-diffuse reaction were found mainly in their peripheral zones.

Succinic dehydrogenase (S. D. H.)

Con trol group: The most intensive reaction was localized in the glandular cellsof the peripheral and central zones. Small graines of the enzymatic reaction productwhich were accompanied by a slightly marked diffuse reaction were localized evenlyin the cytoplasm of hepatocytes (Fig. 13). In the cell of nuclei the reaction was nega­tive.

Experimental group I (30 min): In this gropu a marked increase of the reac­tion intensity was observed mainly in the peripheral zone of the lobules (Fig. 14).

Experimental group II (4 h): In this group no essential differences in charac­ter, localization and intensity were observed as compared with the pictures seen inthe previous group.

Experimental group II (12 h): An increase of the enzymatic reaction was ob­served. Fragments of liver trabecules, filled with very strong grained-diffuse or diffusereaction were seen in the peripheral, intermediate and middle zones (Fig. 15). Hepa­tocytes having vestigial or negative enzymatic reaction were also visible.

Experimental group IV (24 h): In this group the observed histoenzymaticpictures were slightly similar to those seen in the previous group (Fig. 16), but itseems that the number of lobule fragments with negative or very weak reaction wasa bit smaller. The diffuse component of the reaction in the cells of the trabecules withstrong enzymatic reaction was also a little smaller.

Glucose-6-phosphatase (G-6-P-ase)

Control group: Positive reaction for the estimated specific phosphatase oc­cured in all liver lobules. The most intensive reaction was localized in the middlezone of the lobule (Fig. 17, 18). Cell nuclei had no positive reaction (Fig. 23).

Experimental group I (30 min): In this group a slight increase of the reactionintensity was noticed in the intermediate and peripheral zones of the lobules (Fig. 19).

Fig. 13. Positive, fine-grained enzymatic reaction for SDH in the cytoplasm of hepatocytes ofthe controls. X 400.

Fig. 14. Strong diffuse medium, and fine-grained reaction for SDH in adenocytes of the peripheralzone of lobules. I experimental group. X 400.

Fig. 15. Adenocytes of liver filled with very strong grained diffuse or diffuse enzymatic reactionproduct for SDW. III experimental group. X 400

Fig. 16. A fragment of liver lobule with the reaction for SDH IV experimental group. X 400.

Histochemi cal a nd hi stoenzyrnat.ic cha nges

F i ~ . 13 - 16

9

10 M. KAMINSKI et al.

In the liver cells grained-diffuse enzymatic reaction was localized in their cytoplasmlike in the group of healthy animals (Fig. 24).

Experimental group II (4 h): In this group a further increase of the intensityof the colour reaction for G-6-P ase was observed in the peripheral, intermediateand even in the middle zones of the lobules (Fig. 20). In the cytoplasm of the hepato­cytes lying mainly in their peripheral sections, the enzymatic reaction was of coarse­grained-diffuse or diffuse character (Fig. 25), in the remaining ones the localizationand distribution were similar to the pictures of the previous group.

Experimental group III (12 h): A further increase of the reaction intensitywas found in all the zones of the liver lobule (Fig. 21). Few liver cells mainly lying inthe peripheral zone of the lobule had very strong diffuse enzymatic reaction, con­trary to those localized (Fig. 26) in the middle zone that had a weak diffuse reaction.

Experimental group IV (24 h): In the last of the examined. groups, a distinctdecrease in the intensity of the enzymatic reaction was noticed, mainly in the peri­pheral zone of the lobules (Fig. 22). The enzymatic reaction had also a grained-dif­fuse character, and was often localized near the membrane of the nucleus (Figs. 27,28).

Adenosine triphosphatase (A'I'Pvase)

Control group: Positive diffuse reaction was found in the intralobular bilecanaliculi that formed a spatial net, and in the endothelium of the vessels (Fig. 29).The intralobular bile canaliculi were filled with the product of enzymatic reaction,and were arranged in a regular spatial net (Fig. 31).

Experimental group I (30 min): In this group a minimal decrease of the re­action intensity was noticed in the intralobular bile canaliculi. In the endotheliumof the vessels no important differences in character, localization and reaction intensitywere found, as compared with the control group.

Fig. 17. A fragment of the liver of the controle with strong reaction for G-6-P-ase in the middlezone of the lobule. x 100.

Fig. 18. A fragment of the liver of the controls with strong reaction for G-6-P-ase in the middlezone of the lobule. x 100.

Fig. 19. An increase of reaction intensity for G-6-P-ase in the peripheral and middle zone of liver

lobules. I experimental group. X 100.

Fig. 20. A further increase in the reaction intensity for G-6-P-ase in liver lobules. II experimental

group. X 100.

Fig. 21. A further increase of enzymatic reaction for G-6-P-ase in liver lobules. III experimentalgroup. X 100.

Fig. 22. A decrease of reaction intensity for G-6-P-ase in liver lobules. IV experimental group.

X 100.

Histoch emical and h is t oen zy m a t ic ch anges

.Fig. 17- 22

11

12 :\'1. KAMINSKI et al.

Experimental group II (4h): In this group a marked decrease in the inten­sity of the enzymatic reaction was shown in the intralobular bile canaliculi lying inthe peripheral zone of the lobule.

Experimental group III (12 h): The reaction, in comparison with the pre­vious group did not show distinct differences in the intensity, character and locali­zation.

Experimental group IV (24 h): The enzymatic reaction within the intra­lobular bile canaliculi of the periphery of the lobule and in the remaining zones wasa little stronger than in the 2 previous groups. However, some segments of the lobulesshowed a very diffuse reaction for adenosine triphosphatase (Fig. 30). The bile canali­culi in these segments were broken and filled with trace reaction (Fig. 32).

Acid phosphatase (ACP)

Control group: In all liver lobules a positive fine-grained enzymatic reactionwas observed. It was a little stronger in the peripheral zones. In the glandular cellsthe grains were arranged on the secretory poles of hepatocytes along the bile canaliculi(Fig. 33) which, in this way, formed a network. The diameter of these fine graines ofthe reaction was the same as that of lysosomes. In few phagocytes of the BROWICZ­

KUPFFER cells the reaction was strong and grained-diffuse.

Experimental group I (30 min): In the liver cells a considerable increase inthe intensity of the coarse-grained enzymatic reaction with diffuse component wasobserved mainly along the canaliculi (Fig. 34). The grains seemed to be more numer­ous, and their diameter bigger. The increase of grained-diffuse reaction was observedin the border cells too.

Experimental group II (4 h): A slight decrease of the reaction intensity foracid phosphatase was observed as compared with the previous group. It was visibleboth in the peripheral and intermediate zones of the lobule (Fig. 35). Fine and coarse­grained diffuse reaction was found on the secretory poles of the liver cells along theintralobular bile canaliculi (Fig. 35). In very numerous BROWICZ-KuPFFER cells

found in the peripheral zones of the lobules, the enzymatic diffuse reaction was con­

siderably stronger as compared with the previous group.

Fig. 23. Positive grained-diffuse reaction for G-6-P·ase in the cytoplasm of hepatocytes of eon­troIs. X 400.

Fig. 24. Grained-diffuse reaction for G-6-P·ase in adenocytes of I experimental group. X 400.

Fig. 25. Strong coarse grained.diffuse enzymatic reaction for G-6-P·ase in liver cells of II experi­mental group. X 400.

Fig. 26. Single hepatocytes of peripheral zone oflobules with strong diffuse reaction for G-6-P·ase.III experimental group. X 400.

Fig. 27/28. Grained-diffuse enzymatic reaction for G-6-P·ase in adenocytes of the liver in IV ex­perimental group. X 400.

Histochemical and histoenzymatic changes

Fig. 23-28

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14 M. KAMINSKI et al.

Fig. 29-32

Histochemical and histoencymatic changes 15

Experimental group (12 h): In this group a further decrease III the intensityof the grained-diffuse reaction was observed in the intralobular bile canaliculi as com­pared with group II (Fig. 36). In numerous BROWICZ-KuPFFER cells localized mainlyin peripheral zones of the lobules, the reaction was very strong (Fig. 36).

Experimental group IV (24 h): In the peripheral, intermediate as well asmiddle zones of the liver lobules, the activity of the grained reaction for AP in theintralobular bile canaliculi was weaker than in the controls (Fig. 37). In numerous,big BROWICZ-KuPFFER cells a strong diffuse coarse-grained enzymatic reaction fillingthe whole cytoplasm was shown (Fig. 38).

Discussion

The behaviour of colour PAS reaction for glycogen and glucose-6-phosphatase was found tobe the most interesting fact obtained during present investigations. Juxtaposition of these 2 ele­

ments is not accidental, but results from a known functional unity of glucose-6-phosphatase andthis polysaccharide (2, II, 14).

Glucose-6-phosphatase is a specific determinant of endoplasmatic reticulum, and is biochemi­cally responsible for initiation and continuation of glycogenalitic processes, in which glycogenwas used as substratum (2, II, 14). It seems, that the observed marked increase of the reaction

intensity for this specific phosphatase, together with a constant decrease of glycogen in the livercells, initially in the peripheral, and later in the remaining zones, is a convincing argument as tothe functional activition of the endoplasmatic reticulum produced by benzene and its metabolites.

Present investigations as well as the results of our previous· works point to a significant hyper­trophy of endoplasmatic smooth reticulum in adenocytes of both the liver and the kidney. These

results allow the assumption that this very organellum plays a fundamental part in the processesof oxidation and detoxication of benzene (7, 8, 9, 10).

The data from literature indicate that this reticulum is provided with oxidizing enzymes, and

thus may effectively participate in benzene metabolism. On the other hand, the reticulum con­taining glycogen and ebzymatic proteins involved in its decomposition secure normal conjuga­tion of benzene and its metabolites with glucoronic acid (5, 6, 12, 19).

We believe that endoplasmatic smooth reticulum in the liver, and its lipid elements in parti­cular, provide accumulation of benzene and its metabolites in the first place, and then their oxida­tion and glucoronization, due to enzymes contained in its protein walls. The activation of thereaction for glucose-6-phosphatase as well as a decrease of glycogen content in the liver cells arenot even, and mainly concern the fragments of peripheral lobules. Time evolution of this processalso begins from the lobule periphery (30 min group), and ends in the central zone (24 h). It seems

that this rhythm of changes may be due to 2 factors:

1. the peripheral zone of lobules is the most actively reacting part of liver parenchyma to allinternal and external stimuli,

Fig. 29. Positive diffuse reaction for ATP-ase in bile intralobular canaliculi and in endothelium

of liver vessels of controls. X 240.

Fig. 30. Weak enzymatic reaction for ATP-ase in liver lobule of IV experimental group. X 400.

Fig. 31. Strong diffuse reaction for ATP-ase in bile intralobular canaliculi of the liver of controls.

X 400.

Fig. 32. Weak uneven diffuse enzymatic reaction for A'I'Pvase in bile canaliculi of the liver ofIV experimental group. X 400.

16 :\I. E AMIXSKI c t ul .

Fig. 33-38

7 38

Histochemical and histoenzymatic changes 17

2. the lobule circumference primarily contacts with benzene and its metabolites. Thus, theprocesses of deposition, exchanges, as well as toxic effect of benzene in these fragments of lobulestake place first. On the other hand, differentiation of reactions for G-6-P-ase and glycogen amongparticular liver cells of the same zones of lobules are thought to be a manifestation of their func­tional differentiation or focal impairment.

A marked increase (I, II, III experimental groups) of neutral lipids with their consequentdecrease in the last experimental group is an interesting, though difficult to interprete, fact. We

suppose that this increase may be an early signal of accumulation of degenerative changes inthe liver parenchyma. These changes are due to subacute benzene intoxication, though its rapid

normalization in group IV points to reversibility of disturbances in the metabolism and struc­ture of liver cells. Perhaps, this temporary increase of fat content reflects a defective productionof bile by liver cells or difficulty in its secretion.

All metabolic processes, including those of detoxication, taking place in the liver parenchyma

need a supply of a great amount of energy contained in adenosinetriphosphoric acid. Therefore,the behaviour of oxidoreductive enzymes localized in energy producing mitochondria is an im­portant measure of adenocytes efficiency. The behaviour of succinic acid dehydrogenase andNADH2-tetrazol reductase was traced during this experiment. These 2 enzymes that catalizeprocesses of carbohydrate decarboxylation produce electrons, which when passing through eyto­chromal system, bring about charging of ADP to form ATP (3, 21). A marked increase of the dia­

meter and saturation of the granules of enzymatic reaction products along with the appearanceof a strong diffuse component of the reaction is certainly a dominating fact found during the ex­periment. In spite of a seeming increase in the intensity of enzymatic reaction, particularly forDB, the results obtained point to the development of disturbances in tissue respiration. Thus,

the present data are in agreement with former observations in which, by means of electron­microscopy myelin degenerative changes in mitochondria were found (7, 8).

Maximal changes of activity, localization and character of reactions occurring in the zones

of peripheral lobules were also found in mitochondrial enzymes. It seems, that this fact providesanother argument pointing to a particularly heavy loading of these zones by benzene and its

metabolites.Energy production is directly associated with processes of its distribution, whereas their nor­

mal course guarantees constant and efficient course of the synthesis of proteins, carbohydrates,lipids, detoxication and the like. ATP-ase labelled by us is the very enzyme catalizing processesof energy distribution, and at the same time is a measure of the course of an active transport ofsubstances through the cell membrane structures (15).

Fig. 33. Grained enzymatic reaction for acid phosphatase in bile intralobular canaliculi of theliver of controls. X 400.

Fig. 34. Strong medium grained and coarse grained diffuse reaction for ACP in bile intralobular

canaliculi of the liver, of I experimental group. X 400.

Fig. 35. Positive reaction for acid phosphatase in bile intralobular canaliculi of the liver of III ex­

perimental group. X 400.

Fig. 36. Fragment of liver lobule of the lobule periphery with weak reaction for ACP localized

along the course of bile canaliculi and the cytoplasm of the BRowICz-KuPFFER cells with strong

diffusive enzymatic reaction. III experimental group. X 400.

Fig. 37. Weak fine and coarse grained reaction for ACP in bile canaliculi of the liver of IV experi­

mental group. X 400.

Fig. 38. Numerous and giant BROWICZ-KuPFFER cells filled with diffuse enzymatic reaction for

ACP in the peripheral zone of liver lobule of IV experimental group. X 400.

2 Acta histochem. Bd. 61

18 M. KAMINSKI et al.

During subacuts intoxication with benzene this enzyme revealed a marked decrease of in­tensity within bile caniculi in the first 3 experimental groups. However, in group IV a tendency torebuild this activity was slightly marked. Thus, the results obtained may indicate a defectivesecretion of liver cells, resulting either from the impairement of enzyme structure organella orATP synthesis. Acid phosphatase, the last investigated enzyme, is localized within lysosomes

along with a hole group of about 40 enzymes having optimum of activity in an acid pH optimum.These organella, according to data from literature, are determinants of cell catabolism, pha­

gocytic processes, pinocytosis, secretion and excretion (15, 17, 22, 23).In the liver, lyzosomes take an active part in the production of bile, being at the same time

the carriers of its components (16).

The observed increase of enzymatic activity in the secretory pole of hepacytes in the firstexperimental group indicates an increase of catabolic and secretory processes.

In the following time groups (4, 12, 24 h after benzene administration) a decrease of reactionfor AP in the liver cells may be interpreted as a result of a defected synthesis of enzymatic pro­teins, AP included, as well as the damage of lyzosomes.

These changes may probably result in a defected production and secretion of bile. The be­haviour of the reaction for ATP-ase in these groups may provide support for this interpretation.The observed increase in the number and size of the BROWICZ-KuPFFER cells in the subsequenttime groups points to the intensification of the function of the reticulo-endothelial system. Its

role in this case, probably consists in the elimination of the damaged adenocytes.

Conclusions

1. Subacute intoxication with benzene in the liver lobule leads to temporary increase of

neutral lipids content, a decrease of glycogen content, and a number of changes in enzyme activity.2. Constant decrease in the activity of respiratory enzymes provides support for the damage

of the structure and function of mitochondrium.3. A decrease of glycogen amount, and an increase in the activity of glucose-6-phosphatase

indicates that smooth endoplasmic reticulum is involved in the processes of benzene detoxication.4. A decrease in the activity of adenosino-triphosphatase and acid phosphatase, and a tempo­

rary increase of the amount of neutral lipids provide support for the impairment ofstructure andfunction of liver cells.

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Address: Prof. Dr. J. J. JONEK, Department of Morphology, ul. K. Marksa 19, PL - 41-808

Zabrze 8, Poland.

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